Enzymes are used in a variety of commercial applications, including the food and agriculture industry, the pharmaceutical industry, and the chemical and biofuels industry. For example, cellulases, such as endoglucanase, exoglucanase, and β-glucosidase, may be used to convert the cellulosic component of biomass into fermentable sugars for biofuel production. Collectively, these cellulases have been isolated from a broad assortment of organisms, and exhibit a range of temperature, pH, and substrate optima. The cost of cellulases, however, generally presents a barrier to the commercialization of processes using such enzymes.
The costs associated with enzymatic hydrolysis can be lowered by increasing hydrolysis yields or by increasing reactor throughput with the same enzyme loading. Molecular biology methods have been investigated to improve enzyme function and decrease substrate recalcitrance. See Wilson D B., Curr. Opin. Biotech., 2009, 20: 295-299. Alternative methods involving the inclusion of large molecule “additives”, such as surfactants, to hydrolysis mixtures have also been used to increase yields with enzymes. See Eriksson T, et al., Enzyme Microb. Tech., 2002, 31: 353-364; Kaar W E & Holtzapple M T., Biotechnol. Bioeng., 1998, 59(4):419-427; Kristensen J B, et al., Enzyme Microb. Tech., 2007, 40: 888-895; Kumar R & Wyman C E., Biotechnol. Bioeng., 2008, 102(6):1544-1557; Qing Q, et al., Bioresource Technol., 2010, 101:5941-5951; Sipos B, et al., C. R. Biol., 2011, 334: 812-823; and Zheng Y, et al., Appl. Biochem. Biotechnol., 2008, 146:231-248. However, the level at which these additives enhance enzymatic activity can vary based on several factors, including enzyme loading, substrate pretreatment, hydrolysis temperature, and substrate and enzymatic choice. Thus, what are needed in the art are more consistent and reliable methods, viable on an industrial scale that can improve enzymatic activity and stability.
The costs associated with enzymatic hydrolysis can also be lowered by collecting and reusing enzymes through multiple rounds of processing. For example, work has been done in the field of immobilizing cellulases, including their covalent attachment or adsorption onto substrates such as silicon dioxide wafers, silica, glass beads, calcium alginate beads, and magnetic nanoparticles. See e.g., Ogeda, et al., J. Biotechnol. 2012, 157, 246-252; Lupoi, et al., Biotechnol. Bioeng. 2011, 108, 2835-2843; Mandali & Dalaly, ASTM Intern. 2010, 7, 1-10; Andriani, et al., Bioprocess. Biosyst. Eng. 2012, 35, 29-33; Jordan, et al. gala, C. J. Mol. Catal. B-Enzym. 2011, 68, 139-146; Xu, et al., Biocatal. Biotransfor. 2011, 29, 71-76. Work has also been done in the field involving reversibly soluble-insoluble polymer-cellulase materials, most commonly utilizing pH-sensitive polymers such as Eudragit L-100 or methacrylic acid polymers. See e.g., Taniguchi, et al., Biotechnol. Bioeng. 1989, 34, 1092-1097; Liang & Cao, X. Bioresource Technol. 2012, 116, 140-146; Zhang, et al., Biocatal. Biotransfor. 2010, 28, 313-319. These materials, however, limit industrial processes to a fairly narrow pH range and require multiple pH adjustments to recover and reuse the enzyme. Thus, what is also needed in the art are alternatives, viable on an industrial scale, that can reduce enzyme costs by recycling enzymes through multiple rounds of processing.